An adenovirus (Ad) early region 3 (E3) protein called Ad E3-14.7K, which inhibits tumor necrosis factor alpha (TNFalpha) cytolysis, has been used in the yeast two hybrid system to isolate interacting proteins from human cells. The studies were initiated in order to determine the mechanism of action of Ad E3-14.7K. Four proteins called FIPs were detected in a HeLa cell cDNA library. Several of the FIPs interact with RIP (Fas receptor interacting protein), which is an important mediator of cell death induced by TNFalpha and the Fas ligand. FlPI is a novel member of the low molecular weight GTPase superfamily and also has a domain that is homologous to several bacterial proteases. FlPI also associates with a series of cell phosphoproteins following stimulation by TNFalpha and has been used as "bait" to isolate additional cell proteins called GIPs. Several of the GIPs also interact with RIP. Each of the FIPs colocalizes with Ad E3- 14.7K in discrete foci in the cytoplasm. Further studies are proposed to: (1) determine how these various proteins interact physically and functionally in multiprotein complexes. This will be achieved by selected mutagenesis; (2) identify and determine the function of the phosphoproteins that interact with FlP1 and (3) determine if the FlP-1 homologue (ceT24fl) gene in c. elegans affects development. Control of cell death plays an important part in embryonic and fetal development, autoimmunity and cancer as well as other important human diseases. What we learn from studying viral genes that have evolved to prevent cell death by interacting with novel cellular targets should extend our understanding about a variety of human diseases affected by this basic process.